Project Summary Polymicrobial communities that exhibit synergistic pathogenicity cause periodontal disease, one of the most common infections of humans. As a model system for the study of polymicrobial synergy, we are investigating the interactions of Porphyromonas gingivalis and Streptococcus gordonii. Heterotypic communities develop as physiologically compatible organisms modulate their own and their partner's physiology resulting in enhanced fitness and virulence. S. gordonii is an early colonizer of the dental plaque biofilm and provides attachment and support for the more pathogenic, keystone pathogen P. gingivalis. Contact-dependent and soluble metabolite signaling regulates development of the dual species community. Initial development and, ultimately constraint of community development depends upon a tyrosine phosphorylation-signaling cascade. The activities of the kinase and phosphatase, Ptk1 and Ltp1 respectively, signal upstream of the transcriptional regulator CdhR to control gene expression of community effectors such as Mfa1 and LuxS. The objectives of this study are to: i) characterize the structure and function of the Ptk1 kinase activation domain that regulates kinase activity; ii) assess the complex balance of (de)phosphorylation by Ptk1 and Ltp1 that both regulates activity of each protein, while controlling activity of downstream phosphorylation-dependent mediators; and iii) determine the signaling network between tyrosine phosphorylation and CdhR that results in regulation of gene expression. The completion of this study will not only elucidate the mechanism of activation of Ptk1 but also provide critical new insights into the functional activation of BY-kinases. Additionally, we will provide novel information about the dynamic regulation of tyrosine phosphorylation between a BY-kinase and its cognate phosphatase. Finally, we will develop a detailed understanding of the tyrosine phosphorylation cascade that regulates community formation and synergistic pathogenicity between P. gingivalis and S. gordonii. Our overall long-term goal is to translate these novel molecular mechanisms into the development of future therapeutic approaches targeting polymicrobial pathogenic communities. Additionally as BY-kinases are structurally distinct from eukaryotic protein tyrosine kinases, detailed molecular analysis of BY-kinase activation and regulation may provide critical insights for the identification of novel approaches targeting key bacterial signal transduction systems.